sequences encoding their protein. Derocq et al. (2000) conducted studies with wild type and differentiated HL-60 cells, which, as previously mentioned, are derivative of committed neutrophil progenitors of the granulocyte/monocyte lineage (Gallagher et al., 1979), they observed a dramatic decrease of CB2 receptor mRNA transcripts during the course of DMSO-induced differentiation, implying a downregulation of the expressed CB2 receptor. Deusch et al. (2003) were also unable to find any CB2 receptor protein with antihuman CB2 antibodies via chemiluminescent probes. In a similar vein, Oka et al. (2004) using RT-PCR reported that the amount of CB2 receptor mRNA in neutrophils was negligible and that Western blot analysis with an anti-CB2 receptor antibody failed to discern CB2 receptor protein. More recently and in direct contrast to these findings, Kurihara et al. detected cell-surface CB2 expression in both human neutrophils and differentiated neutrophil-like HL-60 cells using flow cytometry. In trying to resolve this conflict, it is worth noting that receptor antibodies are notoriously problematic in the cannabinoid field. Grimsey et al. (2008) conducted a systematic study that found commercially available CB1 antibodies not only fail to detect proteins of the correct molecular weight but also recognize multiple proteins in brain tissues, rendering them ineffective for specific detection of cannabinoid receptors. It is entirely likely that the same is true of commercially available CB2 receptor antibodies, which may be an underlying factor in those studies that failed to detect CB2 expression. In other words, a lack of evidence is not the same as evidence of a lack. Another reasonable explanation arises from the knowledge that CB2 receptor expression in immune cells can be exceptionally plastic and that the specific methods used to prepare the neutrophils may in themselves alter CB2 expression levels (Miller and Stella, 2008). Given the flow cytometry results and the scale of pharmacological data provided by Kuirhara et al. and McHugh et al. , which will be discussed shortly, the weight of evidence currently indicates that human neutrophils do indeed express functional CB2 receptors, although support for CB1 receptors remains sparse.
VII. Inhibition of Induced MiGrATioN: Which REcEpTors are Involved?
Kurihara et al. and McHugh et al. went on to try to elucidate the identity of the receptors the pertinent endocannabinoid ligands activate, by investigating their observed effects in the presence of established CB1, CB2, or TRPV1 antagonists. It is important to note at this point that CB1- or CB2-selective antagonists, such as SR141716A and SR144528, respectively, together with behaving as inverse agonists, can block non-CB1 and non-CB2 targets when administered at concentrations greater than the Kd values determined for their corresponding cannabinoid receptors, that is, at concentrations in the micromolar range (Rinaldi-Carmona et al., 1998; Walter et al., 2003). Kurihara et al. observed that pretreatment of human neutrophils and differentiated HL-60 cells with 1 mM SR144528, but not 1 mM AM251 (a CB1-selective antagonist), attenuated the inhibitory effect produced by 2-AG; however, caution must be exercised in interpreting these results as plainly indicating CB2 receptor involvement. It may well be that CB2 receptors are responsible for transducing the inhibitory signal of 2-AG, but it is also remains a distinct possibility that putative CB2-like cannabinoid receptors mediate the 2-AG effect either alone or in conjunction with CB2. Given the implied scarcity of CB1 expression in neutrophils, a concentration of 1 mM SR141716A was deliberately chosen by McHugh et al. to reduce its antagonism selectivity in the hope of detecting either CB1 or a non-CB1 SR141716A-sensitive receptor. While, 100 nM SR144528 was used in order to ensure its antagonism of CB2 receptors alone. As AEA and NADA are both additionally capable of activating TRPV1 receptors (Ross et al., 2001; Smart et al., 2000; Zygmunt et al., 1999), and although Heiner et al. (2003) have reported that neutrophils do not express TRPV1 receptors, McHugh et al. investigated the inhibitory effect observed in the presence of 1 mM capsazepine (CPZ) to exclude TRPV1 as a receptor candidate.
McHugh et al. tested the ability ofthe two most potent endocannabinoid compounds in the presence ofthe antagonist concentrations specified above and found that the inhibition produced by virodhamine and AEA was significantly attenuated by SR141716A (1 mM); however, the apparent Kb was calculated to be 745.2 nM, which is considerably higher than that expected (~10 nM) for CB1-related antagonism (MacLennan et al., 1998). Surprisingly in the presence of 100 nM SR144528, they observed the inhibition exerted by 100 nM virodhamine and AEA was significantly enhanced rather than attenuated. This enhancement was reproduced in the presence of another CB2-selective antagonist, AM630 (100 nM), to an even greater degree. And lastly, the ability of 100 nM virodhamine and AEA to inhibit fMLP-induced neutrophil migration remained unaffected by 1 mM CPZ, excluding any involvement of TRPV1 receptors as expected. Likewise, SR141716A (1 mM), SR144528 (100 nM), CPZ (1 mM), and AM630 (100 nM) alone had no effect on neutrophil migration triggered by fMLP.
The bulk of evidence gathered indicates that CB1 receptors do not play any role in the cannabinoid-mediated modulation of spontaneous or induced neutrophil migration for the following reasons: there is scant evidence of functional CB1 receptor expression; AEA and virodhamine display potent inhibitory behavior that is inconsistent with their pharmacology at CB1
receptors, that is, AEA acts as a CB1 partial agonist (Mackie et al., 1993; Sugiura et al., 2000), virodhamine as a CB1 antagonist (Porter et al., 2002); 2-AG, which is a full agonist at CB1 receptors (Pertwee and Ross, 2002), in the hands of McHugh et al., had no effect on neutrophil migration, while Kurihara et al. report an inhibitory effect for 2-AG that remains unaffected by the presence of 1 mM AM251; and although the inhibition of fMLP-induced migration observed by McHugh et al. was significantly attenuated by SR141716A, the apparent KB value was inconsistent with a CB1-mediated effect (MacLennan et al., 1998). Taken together, these results are indicative of a role for a non-CB1, SR141716A-sensitive receptor mediating the effect of these cannabinoids.
Kurihara et al. base their case for CB2 receptors transducing the inhibitory signal exerted by 2-AG toward fMLP-induced migration solely upon the observation that this effect is attenuated in the presence of 1 mM SR144528. As noted earlier, it is not possible to confidently assert this interpretation of the data as SR144528 loses its CB2-selectivity in the micromolar concentration range. McHugh et al. imply a role for functional CB2 receptors in the modulation of neutrophil migration on the basis that two, structurally distinct, CB2-selective antagonists, administered at appropriate concentrations, significantly enhanced the cannabinoid-mediated inhibition. There is considerable evidence that cannabinoid CB1 and CB2 receptors exist in a conformation that is precoupled to the G protein (Pertwee, 2005). Both SR144528 and AM630 are CB2 receptor inverse agonists (Bouaboula et al., 1999; Ross et al., 1999), and as such they bind with a high affinity to precoupled CB2 receptors. CB2 receptors on human neutrophils may be constitutively activated, exerting high basal levels of CB2 receptor-mediated signaling; thereby precluding inhibition mediated by certain other receptors. Furthermore, constitutively activation may underlie the lack of significant stimulation of neutrophil migration above basal levels by CB2 agonists, such as 2-AG (Kurihara et al., 2006; McHugh et al., 2008). In line with the findings of McHugh et al., Lunn et al. (2006) have demonstrated that CB2 receptor inverse agonists inhibit leukocyte migration both in vivo and in vitro, and the level of effect is proportional to the degree of inverse efficacy.
Many aspects of the endocannabinoid system have yet to be fully elucidated, and as mentioned previously, a range of putative cannabinoid receptors that exert cannabimimetic activity has been described in the literature. By and large, the reported pharmacology of these receptors is insufficient to account for the array of ligands exerting modulatory activity on spontaneous or induced neutrophil migration observed by Kurihara ei a/. and McHugh ei a/. The exception being the non-CBj, non-CB2 pharamcological target named the Abn-CBD receptor, which is antagonized by SR141716A at concentrations considerably higher than those predicted from its CB1 receptor affinity (Begg ei a/., 2005). Evidence for the existence of Abn-CBD receptors initially emerged from studies in certain blood vessels from CB1_/_ mice (Jarai ei a/., 1999). In addition, virodhamine and NADA induce a relaxation of mesenteric arteries, being more potent than either AEA or Abn-CBD (Begg ei a/., 2005; Ho and Hiley, 2003; O'Sullivan ei a/., 2006). Probably of greatest relevance are studies in microglial cells, which provide robust evidence for a role of Abn-CBD receptors in cell migration (Walter ei a/., 2003). Thus, 2-AG triggers microglial cells by acting through CB2 and Abn-CBD receptors. Stimulation of microglial cell migration by cannabinoids is antagonized by high but not low concentrations of SR141716A (Franklin and Stella, 2003). Certain parallels exist between the pharmacology that we observed in human neutrophils and the pharmacology of the Abn-CBD receptor. First, the effects of both AEA and virodhamine are sensitive to antagonism by 1 mM SR141716A, which is in line with the proposed affinity of the CB1 receptor antagonist for the Abn-CBD receptor (Begg ei a/., 2005). Second, agonist efficacy and potency closely match that previously obtained for the Abn-CBD receptor in blood vessels and microglial cells; of the endocannabinoids and related lipids, AEA, NADA, and virodhamine are active, the latter having the highest potency, while PEA is inactive. McHugh ei a/. also report the antagonism of endocannabinoid-induced inhibition by N-arachidonoyl-L-serine (ARA-S), a brain constituent previously shown to be an agonist at the Abn-CBD receptor (Milman ei a/., 2006). They report that this endogenous compound attenuates the inhibition of human neutrophil migration by AEA, virodhamine, and Abn-CBD. Although 1 mM ARA-S abolishes the effect of Abn-CBD completely, it reduces the Emax value of virodhamine, implying that the effect of virodhamine may involve more than one receptor, only one ofwhich is blocked by ARA-S. Alternatively, ARA-S may be acting allosterically on the target receptor; a reduction in Emax is characteristic of an allosteric inhibitor. In line with these findings, in a recent abstract Zhang ei a/. (2005) report that Abn-CBD inhibits angiogenesis, an effect that is antagonized by ARA-S. This raises two possibilities: either this endogenous lipid mediator is a partial agonist at the Abn-CBD receptor and thereby also acts as an antagonist in certain conditions; or the effects observed by McHugh ei a/. are due, at least in part, to activation of another, perhaps related target.
Recent publications (Johns ei a/., 2007; Ryberg ei a/., 2007) have emerged suggesting certain cannabinoid ligands interact with the orphan receptor GPR55 and that this, and possibly other orphan receptors, may account for the pharmacological and functional evidence for some novel cannabinoid receptors (Baker et al., 2006; Pertwee, 2007). Given the possible relevance of orphan GPCRs with regard to migratory signaling, it is pertinent to discuss any potential involvement ofthis novel cannabinoid receptor in the modulation of neutrophil migration. GPR55 was cloned by Sawzdargo et al. (1999) and maps to chromosome 2q37; it is poorly related to other receptors, sharing closest identity with the orphan receptors, GPR35 (37%) and GPR92 (30%), and 13.5 and 14.4% homology with CB1 and CB2 receptors, respectively (Baker et al., 2006). The expression profile ofGPR55 has been reported to be lung > spleen > kidney > brain > heart (Brown and Wise, 2001). Additionally, it has been located in smaller mesenteric arteries ofhuman colon tissue and is highly expressed in adipose (Brown et al., 2005; Drmota et al., 2004). Some controversy surrounds the pharmacology of GPR55; two pharmaceutical companies, AstraZeneca plc and GlaxoSmithKline Inc, have independently demonstrated that GPR55 can be activated by endogenous, phyto and synthetic cannabinoid ligands (Brown and Wise, 2001; Drmota et al., 2004). However, the pharmacology of GPR55 reported by AstraZeneca and GlaxoSmithKline is somewhat conflicting; moreover, Oka et al. (2007) report that LPI, but not cannabinoid ligands, induces extracellular signal-related kinase (ERK) phosphorylation in GPR55-expressing cells.
Most pertinent to the discussion ofendocannabinoid-induced inhibition of neutrophil migration is the suggestion that the Abn-CBD receptor is, in fact, GPR55; however, there are substantial differences in the reported pharmacology of these two targets, which argues against this being the case (Baker et al., 2006; Mackie and Stella, 2006). Furthermore, although Abn-CBD has high affinity for GPR55, it retains its vasodilator effects in GPR55-/~ mice, the implication being that this atypical cannabinoid activates more additional novel receptors (Hiley and Kaup, 2007; Johns et al., 2007).
That said and with regard to human neutrophils, it is relevant that GPR55 is expressed in splenic tissue and that virodhamine, which was the highest efficacy and potency compound in the study by McHugh et al., is also reported to be a high-efficacy agonist of GPR55 (Ryberg et al., 2007). Kurihara et al. have demonstrated that their CB2-like receptor plays a role in human neutrophil migration by modulating RhoA activation, which will be discussed shortly. McHugh et al. report that CBD potently inhibits, while LPI does not inhibit human neutrophil migration, which is in conflict with the reported pharmacology of GPR55 by Oka et al. (2007) and Whyte et al. (2008). Reports suggest that GPR55 and related orphan receptors are G13-coupled and that they can activate the Rho pathway (Henstridge et al., 2008; Ryberg et al., 2007), which plays an important role in the regulation of myosin light chain phosphorylation and subsequent cytoskeletal-dependent locomotion (Buhl et al., 1995; Kozasa et al., 1998). Given such G
protein-coupling, as shall be discussed shortly, agonists at GPR55 would be expected to stimulate neutrophil migration rather than inhibiting it. Currently, further work is required to establish the molecular identity of the Abn-CBD receptor.
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